West Nile virus, a member of the flavivirus family of positive strand RNA viruses, has spread rapidly across North America since its introduction in the summer of 1999. This summer of 2002 has brought an epidemic of surprising ferocity, with over two thousand human cases and nearly 100 deaths recorded by late September. The virus is expected to be endemic in all U.S. mainland states by some time next year. The goal of this proposal is to initiate experiments aimed at understanding the regulation of West Nile viral gene expression, which occurs by translation of the genomic RNA that is unusual in possessing a 5'-cap but lacking a 3'-poly(A) tail. This R03 proposal represents the beginning of work on West Nile virus by an investigator experienced with studies on other positive strand RNA viruses of similar genome design. The proposal has two focusses. The first aim is to pinpoint domains in the 5' and 3' terminal regions of the viral genomic RNA that are involved in directing its translation by host ribosomes. The basic information obtained will be the springboard for future detailed studies of interactions between the viral RNA and the host cell's translation initiation factors. Such studies are expected to lead to a molecular understanding of the regulation of viral gene expression, the first event of the viral lifecycle after the virus gains access to a host cell. The second aim is to determine whether West Nile virus induces a host RNA-interference (RNAi) response, and whether the virus encodes a protein specifically designed to suppress that response. If a virus-specific RNAi response occurs in host cells, successful viral gene expression would require interdiction of the response. Though well studied with plant viruses, the involvement of RNAi in the virus/host interplay is in its infancy with animal viruses. Investigating the relevance of RNAi to West Nile virus infections is considered an innovative feasibility study that could lead to an important breakthrough and more detailed exploration in a subsequent proposal. Our studies will contribute new information useful for the design of protective strategies against West Nile virus, for which no vaccine is currently available for human use. Our studies will also be applicable to other flaviviruses for which there is at present no vaccine, including dengue virus.